Method for producing liposome and apparatus for producing liposome

ABSTRACT

Disclosed herein are a method for producing a liposome which is capable of reducing the facility costs and also capable of rapid desolvation, and an apparatus for producing a liposome which is for use in the above-mentioned method. Provided is a method for producing a liposome, including a stirring step of stirring a mixed liquid containing an oil phase in which at least one lipid is dissolved in an organic solvent and a water phase, and an evaporating step of evaporating an organic solvent from the mixed liquid, in which the condensed organic solvent is removed by passing a gas having a temperature not higher than the dew point of the solvent in the evaporating step.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of co-pending U.S. patentapplication Ser. No. 15/625,020 filed Jun. 16, 2017, which is acontinuation of International Application PCT/JP2015/085456 filed onDec. 18, 2015, which claims priority under 35 U.S.C. § 119 of JapanesePatent Application No. 257283/2014 filed on Dec. 19, 2014, all of whichare hereby expressly incorporated by reference into the presentapplication

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a method for producing a liposome andan apparatus for producing a liposome. Specifically, the presentinvention relates to a method for producing a liposome which can besuitably used for pharmaceutical applications and an apparatus forproducing such a liposome.

2. Description of the Related Art

A liposome (hereinafter, also referred to as lipid vesicle) is a closedvesicle formed of a lipid bilayer membrane using lipids, and has anaqueous solution (outer water phase) in which liposomes are dispersedand a water phase (inner water phase) within the space of the closedvesicle. Liposomes have been studied for a variety of applications suchas immune sensors, artificial red blood cells, and carriers of drugdelivery systems taking advantage of the features such as barriercapacity, compound retention capacity, biocompatibility, the degree offreedom of setting the particle size, ready biodegradability, andsurface-modifying properties. As for the application of a carrier,liposomes can encapsulate water-soluble compounds, lipophiliclow-molecular weight and high-molecular weight materials, and a widerange of materials. As an example of the method for producing aliposome, there is a method in which a water phase and an oil phase arestirred and emulsified in a stirring apparatus, followed by desolvation.

JP2005-505645A (paragraph 0074) discloses that an emulsion is preparedby stirring a dispersion system in which an oil based solution A isadded to an aqueous solution B, flushing the dispersion system withnitrogen, and then maintaining the dispersion system at 55° C. for 60minutes to evaporate the residual methylene chloride. WO01/083594A(Example 9) discloses that an emulsion is poured into a cylindricalairtight container and stirred at room temperature at 400 rpm while anitrogen gas is simultaneously passed through the hollow fiber for onehour to remove methylene chloride from the container, thereby obtainingmicrosphere particles. JP2010-285438A (paragraph 0149), JP1997-503225A(JP-H09-503225A) (claim 4), and JP1990-502460A (JP-H02-502460A) alsodisclose that the produced emulsion is subjected to removing of asolvent. Similarly, JP1989-009931A (JP-H01-009931A) (Example 3),JP2002-516260A (Example 4), JP2013-529677A (paragraph 0103), andJP2001-505224A (Example 3) also disclose that the produced emulsion issubjected to removing of a solvent.

SUMMARY OF THE INVENTION

In the preparation of liposomes, it is important to rapidly remove thesolvent in the liquid by a desolvation step in order to preventre-coalescence of the dispersion miniaturized in an emulsifying step andto stabilize the particle size. In order to achieve the desolvation, itis necessary to supply heat from an external by a hot water jacket orthe like. However, if the liquid preparation scale becomes larger, theheat transfer area per volume of the liquid becomes smaller, so the heatsupply becomes insufficient and the evaporation rate will decrease. As aresult, the re-coalescence of the dispersion proceeds and therefore itbecomes impossible to reduce the particle size of the liposomes.

In order to achieve rapid desolvation, it is necessary to discharge theevaporated solvent gas and water vapor to the exterior of thedesolvation vessel in a gaseous state at a rapid rate. For this purpose,however, a large amount of clean air or nitrogen (carrier gas) isrequired and the capital investment burden to supply them becomesexcessive, which was an obstacle to practical use. In order to reducethe amount of carrier gas being used, it is necessary to heat thecarrier gas to thereby increase the saturated vapor volume. However, ithas not been possible to solve the problem because it was necessary toinvest in the heating equipment of the carrier gas.

It is an object of the present invention to provide a method forproducing a liposome which is capable of reducing the facility costs andalso capable of rapid desolvation, and an apparatus for producing aliposome for use in the above-mentioned method.

As a result of extensive studies to solve the above-mentioned problems,the present inventors have found that, in a method for producing aliposome, including a stirring step of stirring a mixed liquidcontaining an oil phase and a water phase and an evaporating step ofevaporating an organic solvent from the mixed liquid, desolvation can berapidly achieved by passing a gas at a temperature not higher than thedew point of the solvent in the evaporating step to thereby remove thecondensed organic solvent. The present invention has been completedbased on such findings. That is, according to the present invention, thefollowing will be provided.

(1) A method for producing a liposome, comprising a stirring step ofstirring a mixed liquid containing an oil phase in which at least onelipid is dissolved in an organic solvent and a water phase, and anevaporating step of evaporating an organic solvent from the mixedliquid, in which the condensed organic solvent is removed by passing agas having a temperature not higher than the dew point of the solvent inthe evaporating step.

(2) The method for producing a liposome according to (1), in which thestirring step and the evaporating step are carried out in a tank, andthe ratio A/B of length A of the widest portion of the liquid horizontalplane of the mixed liquid in the tank at the start of the evaporatingstep to length B of the deepest portion of the liquid depth is 2 ormore.

(3) The method for producing a liposome according to (1) or (2), inwhich the stirring step and the evaporating step are carried out in atank, and the ratio C/D of length C of the widest portion of thehorizontal plane of the gas space in the tank at the start of theevaporating step to length D of the longest portion of the height of thegas space is 3 or more, where the gas space refers to a space occupiedby the gas flowing in a horizontal direction on the liquid surface.

(4) The method for producing a liposome according to any one of (1) to(3), in which the capacity of the tank is 10 L or more and 100 L orless.

(5) The method for producing a liposome according to any one of (1) to(4), in which the evaporating step is carried out by stirring using acentrifugal stirrer.

(6) The method for producing a liposome according to any one of (1) to(5), in which the centrifugal stirrer has 2 to 10 discharge ports, thedischarge rate coefficient indicated by total opening area of thedischarge ports×circumferential length is 60 cm³ to 6,000 cm³, and therotation speed of the centrifugal stirrer is 100 to 1,500 rpm.

(7) The method for producing a liposome according to any one of (1) to(6), in which in the evaporating step, the gas is sucked into thecontainer from the central portion of the upper portion of thecontainer, and the gas and the condensed organic solvent are dischargedfrom the peripheral portion of the upper portion of the container.

(8) An apparatus for producing a liposome, comprising a tank, a stirrer,and a jacket, in which the cross-section of the space in the tank has acircular shape, the size of the circle in the cross-section is variabledepending on the height of the tank, the stirrer is inserted into thetank from the center of the lid at the top of the tank, the jacket isprovided around the tank to control the temperature in the tank, one ormore inlet ports for sucking gas into the tank are provided at thecentral portion of the lid at the top of the tank, and one or moreoutlet ports for discharging the gas and condensed organic solvent fromthe tank are provided at the peripheral portion of the lid at the top ofthe tank.

(9) The apparatus for producing a liposome according to (8), in whichthe capacity of the tank is 10 L or more and 100 L or less.

According to the present invention, it is possible to reduce the usedamount of carrier air (nitrogen or clean air) required for desolvationand reduce the cost of the equipment. Further, according to the presentinvention, it is possible to achieve efficient heat balance and rapiddesolvation by recovering the latent heat of evaporation by mistgeneration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a configuration example of a centrifugal stirrer.

FIG. 2 shows a top view of a configuration example of an apparatus forproducing a liposome according to the present invention.

FIG. 3 shows a cross-sectional view of a configuration example of anapparatus for producing a liposome according to the present invention.

FIG. 4 shows a configuration of an emulsifying apparatus used in theExamples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “step” used herein includes not only an independent step, butalso a step which may not be clearly separated from another step,insofar as an expected effect of the step can be attained.

The numerical ranges shown with “to” in the present specification meansranges including the numerical values indicated before and after “to” asthe minimum and maximum values, respectively.

In referring herein to a content of a component in a composition, in acase where plural substances exist corresponding to a component in thecomposition, the content means the total amount of the plural substancesexisting within the composition, unless otherwise specified.

Hereinafter, the present invention will be described in detail.

The method for producing a liposome according to the present inventionis a method for producing a liposome, including a stirring step ofstirring a mixed liquid containing an oil phase in which at least onelipid is dissolved in an organic solvent and a water phase, and anevaporating step of evaporating an organic solvent from the mixedliquid, in which the condensed solvent is removed by passing a gashaving a temperature not higher than the dew point of the solvent in theevaporating step.

As a basic idea of the drying technique and the desolvation technique,it is a principle to discharge the evaporated solvent gas and watervapor out of the system by blowing a carrier gas in an amount and at atemperature sufficient to maintain such evaporated solvent gas and watervapor below a saturated vapor volume thereof. There is no known systemfor desolvation under the conditions above the saturated vapor volume.Therefore, the method for producing a liposome according to the presentinvention, which includes the removal of the condensed organic solventby passing a gas having a temperature not higher than the dew point ofthe solvent, is based on the fact that the idea itself is novel. Thereason why the removal of the condensed organic solvent has not beenconceived until now is thought to be due to the fact that there was aconcern that the efficiency of desolvation would be significantlylowered because the condensed solvent or water would turn into waterdroplets and be refluxed onto the inner wall of the container or exhaustair duct if it was not discharged quickly out of the container. Asdescribed above, the idea of the present invention is contradictory tothe basic conventional principle, and thereby achieving rapiddesolvation is a completely unexpected advantageous effect.

That is, according to the present invention, it is possible to dischargethe solvent out of the system with a smaller amount of carrier gas, dueto it not being restricted by the saturated vapor pressure by physicallydischarging a portion or all of the solvent gas and water vapor on acarrier gas while condensing (mist generation) at a dew point or less.In this case, the latent heat absorbed once the solvent and water areevaporated is recovered by the heat generated during the mistgeneration, so that rapid desolvation can be achieved by a smaller heatsupply of the jacket. Further, the heating equipment of the carrier gasalso becomes unnecessary.

(Liposome)

Liposome is a lipid vesicle formed from a lipid bilayer containing lipidmolecules. Specifically, the liposome refers to a vesicle containing aclosed lipid having a space separated from the external environment by alipid bilayer formed based on the polarity of the hydrophobic groups andhydrophilic groups of the lipid molecules. The liposome is a closedvesicle formed of a lipid bilayer membrane using lipids, and has a waterphase (inner water phase) within the space of the closed vesicle. Theinner water phase contains water and the like. The liposome may besingle lamellar (monolayer lamellar, unilamellar, or single bilayermembrane) or multilayered lamellar (multilamellar, which is anonion-like structure having multiple bilayer membranes where individuallayers are compartmented by water-like layers). In the presentinvention, a monolayer lamellar liposome is preferred from the viewpointof safety and stability in pharmaceutical applications.

The liposome is preferably a liposome capable of encapsulating a drugand is not particularly limited in terms of form. The “encapsulating”means taking a form in which a drug is contained in an inner water phaseand a membrane with respect to the liposome. For example, the liposomemay be a form where a drug is encapsulated within a closed space formedof a membrane, a form where a drug is included in the membrane itself,or a combination thereof.

The size (average particle size) of the liposome is not particularlylimited and is preferably 100 nm or less, more preferably 30 to 70 nm,still more preferably 40 to 60 nm, and particularly preferably 40 to 55nm. The liposome is preferably in the form of a spherical shape or ashape close thereto. In the present invention, the “average particlesize” means a volume average value of the diameters of liposomes asmeasured by a dynamic light scattering method.

The component (membrane component) constituting the lipid bilayer of aliposome is selected from lipids. As the lipid, any one may be used aslong as it is dissolved in a mixed solvent of a water-soluble organicsolvent and an ester-based organic solvent. Specific examples of lipidsinclude phospholipids, lipids other than phospholipids, cholesterols andderivatives thereof. These components may be composed of single orplural components.

Examples of the phospholipid include natural or synthetic phospholipidssuch as phosphatidylcholine (lecithin), phosphatidyl glycerol,phosphatidic acid, phosphatidylethanolamine, phosphatidyl serine,phosphatidyl inositol, sphingomyelin, and cardiolipin, or hydrogenatedproducts thereof (for example, hydrogenated soybean phosphatidylcholine(HSPC)). Among these, preferred is a hydrogenated phospholipid such ashydrogenated soybean phosphatidylcholine, or sphingomyelin, and morepreferred is hydrogenated soybean phosphatidylcholine. In the presentinvention, the “phospholipid” also encompasses a phospholipid derivativein which the phospholipid is modified.

Lipids other than phospholipids may be lipids containing no phosphoricacid, and examples thereof include, but are not particularly limited to,glycerolipid which does not contain a phosphoric acid moiety within themolecule, and sphingolipid which does not contain a phosphoric acidmoiety within the molecule. In the present invention, the term “lipidsother than phospholipids” also encompasses derivatives of lipids otherthan phospholipids in which modifications have been made to the lipidsother than phospholipids.

In the case where the lipids other than phospholipids contain a basicfunctional group, for example, in the case where the lipids other thanphospholipids are a material where a compound having a basic functionalgroup is bonded to a lipid, the lipid is referred to as a cationizedlipid. The cationized lipid becomes capable of modifying, for example,the membrane of the liposome and therefore can enhance the adhesivenessto cells which are the target sites.

Examples of cholesterols include cholesterol which containscyclopentahydrophenanthrene as a basic skeleton whose carbon atoms arepartially or completely hydrogenated and derivatives thereof. Specificexamples of cholesterols include, but are not particularly limited to,cholesterol. When the average particle size decreases to 100 nm or less,the curvature of the lipid membrane becomes higher. Since thedeformation of the membrane arranged within the liposome also increases,a water-soluble drug becomes more susceptible to leakage. However, as ameans for suppressing leakage properties, it is effective to addcholesterol or the like in order to fill the deformation of the membranecaused by the lipid (membrane-stabilizing effect).

The addition of cholesterol into liposome compositions is expected tolower the fluidity of the liposome membrane by filling membrane gaps ofliposomes, or the like. Generally, it has been desired that the contentof cholesterol in liposomes is usually an amount of up to about 50 mol %of the total moles of lipid components (total lipid).

In the present invention, in total moles of lipid components in theliposome composition (total lipid contained in the liposomecomposition), the content of cholesterol is preferably 10 to 35 mol %,more preferably 15 to 25 mol %, and still more preferably 17 to 21 mol%.

In addition to the above-mentioned components, a hydrophilic polymer orthe like for improving retentivity in blood, fatty acid, diacetylphosphate or the like as a membrane structure stabilizer, orα-tocopherol or the like as an antioxidant may be added to the liposome.In the present invention, it is preferable not to use additives such asa dispersing aid not approved for use as an intravenous injection inpharmaceutical applications, for example, a surfactant or the like.

The liposome preferably contains hydrophilic polymer-modified productsof phospholipids, lipids other than phospholipids, or cholesterols asphospholipids, lipids other than phospholipids, cholesterols andderivatives thereof.

Examples of the hydrophilic polymer include, but are not particularlylimited to, polyethylene glycols, polyglycerols, polypropylene glycols,polyvinyl alcohols, a styrene-maleic anhydride alternating copolymer,polyvinylpyrrolidone, and synthetic polyamino acid. The above-mentionedhydrophilic polymers may be used alone or in a combination of two ormore thereof.

Among these, from the viewpoint of retentivity in blood of aformulation, preferred are polyethylene glycols, polyglycerols, andpolypropylene glycols, and more preferred are polyethylene glycol (PEG),polyglycerol (PG), and polypropylene glycol (PPG). Polyethylene glycol(PEG) is most commonly used and is preferable due to having an effect ofimproving the retentivity in blood.

The molecular weight of PEG is not particularly limited. The molecularweight of PEG is 500 to 10,000 daltons, preferably 1,000 to 7,000daltons, and more preferably 2,000 to 5,000 daltons.

In the liposome, it is preferable to use a lipid modified by PEG(PEG-modified lipid), together with the main lipid contained in theliposome. Specific examples of the PEG-modified lipid include1,2-distearoyl-3-phosphatidylethanolamine-PEG (DSPE-PEG; manufactured byNippon Oil & Fats Co., Ltd.; specifically1,2-distearoyl-3-phosphatidylethanolamine-PEG2000, or1,2-distearoyl-3-phosphatidylethanolamine-PEG5000 is preferable) anddistearoyl glycerol-PEG2000 (manufactured by Nippon Oil & Fats Co.,Ltd.). These PEG-modified lipids may be added in an amount of 0.3 to 50mass %, preferably 0.5 to 30 mass %, and more preferably 1 to 20 mass %with respect to total lipid content.

In the liposome, preferred is a lipid combination of hydrogenatedsoybean phosphatidylcholine (a main lipid contained in the liposome) and1,2-distearoyl-3-phosphatidylethanolamine-PEG (a lipid used incombination with the main lipid).

The liposome preferably does not contain an anionic polymer (polyanion).In the present invention, since it is possible to control the releaseability by means of an osmotic pressure of an inner water phase, thereare advantages in that general versatility is excellent, and drugs whichcan be used in liposomes are not limited.

(Method for Producing Liposome)

The method for producing a liposome according to the present inventionis a method for producing a liposome, including a stirring step ofstirring a mixed liquid containing an oil phase in which at least onelipid is dissolved in an organic solvent and a water phase, and anevaporating step of evaporating an organic solvent from the mixedliquid, in which the condensed organic solvent is removed by passing agas having a temperature not higher than the dew point of the solvent inthe evaporating step.

The emulsifying step of emulsifying mixed lipids dissolved in an organicsolvent to form a liposome, without a drying and solidifying step, isnot limited as long as it is a step of emulsification, but it ispreferably a step of applying a high shearing force and performingmicroparticulation in an emulsifying step including an organic solvent.If necessary, evaporation (desolvation) of the organic solvent used inthe emulsifying step may be carried out to form a liposome.

(Oil Phase)

As the organic solvent serving as an oil phase, a mixed solvent of awater-soluble organic solvent and an ester-based organic solvent isused. In the present invention, it is preferred that an organic solventsuch as chloroform, methylene chloride, hexane, or cyclohexane is notsubstantially used as the organic solvent, and it is more preferred thatthese organic solvents are not used at all.

The water-soluble organic solvent is not particularly limited, and it ispreferably an organic solvent having a property that is spontaneouslymiscible with water. Specific examples of the water-soluble organicsolvent include alcohols such as methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, and t-butanol; glycols such asglycerin, ethylene glycol, and propylene glycol; and polyalkyleneglycols such as polyethylene glycol. Among these, preferred arealcohols. The alcohol is preferably at least one selected from ethanol,methanol, 2-propanol, or t-butanol, more preferably at least oneselected from ethanol, 2-propanol, or t-butanol, and still morepreferably ethanol.

The ester-based organic solvent is not particularly limited, and it ispreferably an ester obtained from the reaction of organic acids andalcohols. Specifically, the ester-based organic solvent is preferably atleast one selected from ethyl acetate, methyl acetate, isopropylacetate, t-butyl acetate, or methyl propionate, more preferably ethylacetate, isopropyl acetate, or methyl propionate, and still morepreferably ethyl acetate.

The mixing ratio of water-soluble organic solvent:ester-based organicsolvent is not particularly limited, and it may be about 90:10 to 20:80and preferably about 70:30 to 25:75 by mass ratio. The mixed solvent ofa water-soluble organic solvent and an ester-based organic solvent mayfurther contain an aqueous solvent to be described below, such as wateror buffer. The aqueous solvent may be added in a range of, for example,1 to 30 mass %. The pH of the mixed solvent is not particularly limited,and it is in the range of preferably about 4 to 10 and more preferablyabout 6 to 9. The ester-based organic solvents may containphysiologically active substances or the like such as various drugswhich are soluble in these solvents.

The concentration of the lipid is not particularly limited and may beappropriately adjusted, but it may be 40 g/L to 250 g/L, preferably 100g/L to 200 g/L in terms of a solution where a mixed liquid of awater-soluble organic solvent and an ester-based organic solvent servesas a solvent.

(Water Phase)

As a water phase (aqueous solution) where the membrane components aredispersed when producing liposomes, water (distilled water, water forinjection, or the like), physiological saline, various buffers, anaqueous solution of sugars or a mixture thereof (aqueous solvent) ispreferably used. The buffer is not limited to organic and inorganicbuffer solutions, and a buffer having a buffering action in the vicinityof a pH close to that of a body fluid is preferably used and examplesthereof include phosphate buffer, Tris buffer, citrate buffer, acetatebuffer, and Good's buffer. The pH of the water phase is not particularlylimited, and it may be 5.0 to 9.0 and preferably 7.0 to 8.0. Forexample, a phosphate buffer (for example, pH=7.4) is preferably used.The aqueous solution finally contained in the inner water phase of theliposome may be an aqueous solution for dispersing the membranecomponents when producing the liposome, or may be water, physiologicalsaline, various buffers, an aqueous solution of sugars or a mixturethereof which is newly added. The water used as a water phase (aqueoussolution) is preferably free from impurities (dust, chemicals, or thelike).

(Emulsifying Step)

In the emulsifying step, an oil phase where at least one lipid has beendissolved in an organic solvent and a water phase are mixed to preparean aqueous solution containing lipids, which is then emulsified bystirring. An oil phase where lipids have been dissolved in an organicsolvent and a water phase are mixed, stirred and emulsified to therebyprepare an emulsion where an oil phase and a water phase are emulsifiedin an O/W type. After mixing, a liposome is formed by removing a portionor all of the organic solvent derived from the oil phase using anevaporating step to be described below. Alternatively, a portion or allof the organic solvent in the oil phase is evaporated during the courseof the stirring-emulsification to form a liposome.

As a method of stirring, ultrasonic waves or mechanical shearing forceis used for particle miniaturization. In addition, extruder processingof allowing to pass through a filter having a certain pore size ormicrofluidizer processing may be carried out for uniformity of theparticle sizes. Use of an extruder or the like can result in deformationof secondarily formed multivesicular liposomes into univesicularliposomes. In the present invention, it is preferred from the viewpointof simplification of the production process that a liposome of a statethat has no drug loading is used in the next step without extrusionprocessing.

Emulsification may be carried out by stirring in a tank. The capacity ofthe tank is not particularly limited, but from the viewpoint of largescale production being favorable, it is preferably 10 L or more and 100L or less and more preferably 20 L or more and 100 L or less.

When carrying out the emulsification, a mixture can be circulated usinga pump, which may be a turbo-type pump (a centrifugal pump, a diagonalflow pump, or an axial flow pump), a positive displacement reciprocatingpump (a piston pump, a plunger pump, or a diaphragm pump), a positivedisplacement rotary pump (a gear pump, a vane pump, or a screw pump), atube pump, or the like. Among them, a tube pump or diaphragm pump havingno sliding surfaces may be preferably used. Particularly preferred is adiaphragm pump that can easily secure a large flow rate.

For emulsification, it is preferable to stir a mixture in a tank usingan emulsifying apparatus. Examples of the emulsifying apparatus that canbe used include an impeller type, a sawtooth blade type, a closed rotortype, a rotor/stator type, a static mixer type, a colloid mill type, anda high-pressure homogenizer type. Among them, preferred is an impellertype, a sawtooth type, a closed rotor type, or a rotor/stator type whichis suitable for batch processing in a tank.

Particularly preferred is a rotor/stator type which is capable ofgenerating a jet flow in a treatment liquid by high-speed rotation forminiaturization, and capable of sufficiently exerting shear forcesbetween liquid and liquid or liquid and wall surface. As an example, anintermittent jet flow generating type emulsifying apparatus can be used.

In the present invention, an average particle size of a liposome to beprepared can be controlled by arbitrarily selecting the speed and timeof stirring. In view of obtaining a liposome having safety andstability, it is preferable to provide shearing at a circumferentialspeed of 15 m/sec or higher to an aqueous solution containing lipids.Shearing is not limited, and a specific example thereof is shearing at acircumferential speed of preferably 15 m/sec or higher and 35 m/sec orlower, more preferably shearing at a circumferential speed of 20 m/secor higher and 35 m/sec or lower, and still more preferably shearing at acircumferential speed of 23 m/sec or higher and 30 m/sec or lower.

(Evaporating Step)

The method of the present invention includes an evaporating step ofevaporating an organic solvent from a mixed liquid of the stirring step.In the evaporating step, the organic solvent is evaporated from theaqueous solution containing the liposomes obtained in the emulsifyingstep. The evaporating step includes at least one of a step of forciblyremoving a portion or all of the organic solvent derived from the oilphase as an evaporating step and a step of naturally evaporating aportion or all of the organic solvent in the oil phase during the courseof stirring-emulsification.

The method of evaporating an organic solvent in the evaporating step isnot particularly limited as long as it is a method of removing thecondensed organic solvent by passing a gas having a temperature nothigher than the dew point of a solvent. The solvent in “dew point of asolvent” means the total solvent in a mixed liquid, including an organicsolvent and water.

Preferably, the stirring step and the evaporating step are carried outin a tank, and the ratio A/B of length A of the widest portion of theliquid horizontal plane of the mixed liquid in the tank at the start ofthe evaporating step to length B of the deepest portion of the liquiddepth is 2 or more, more preferably 3 or more, still more preferably 3.5or more, and particularly preferably 4 or more.

Preferably, the stirring step and the evaporating step are carried outin a tank, and the ratio C/D of length C of the widest portion of thehorizontal plane of the gas space in the tank at the start of theevaporating step to length D of the longest portion of the height of thegas space is 2 or more, more preferably 3 or more, still more preferably3.5 or more, and particularly preferably 4 or more at the start ofdesolvation.

The gas space as used herein refers to a space occupied by the gasflowing in a horizontal direction on the liquid surface. That is, theheight of the space may exhibit a local height variation due to theshape of the lid at the top of the tank container. The term “spaceoccupied by the gas flowing in a horizontal direction on the liquidsurface” refers to a space occupied by the gas in air flow state,without taking into account such a local height variation.

A sufficient heat transfer area and a sufficient gas-liquid interfacialarea can be ensured by setting A/B to 2 or more and/or C/D to 2 or moreas described above, so that it becomes possible to achieve a more rapiddesolvation.

Preferably, the evaporating step may be carried out while stirring usinga centrifugal stirrer. In order to achieve rapid desolvation, it iseffective to improve a heat transfer coefficient. As a means for thispurpose, it is considered to strengthen the stirring. However, if thestirring is strengthened, air bubbles will be generated by airentrainment. In particular, in the case of using a tank having A/B of 2or more and/or C/D of 2 or more, the water depth becomes shallow andconsequently air entrainment becomes severe. It has now been found thatthe use of a centrifugal stirrer makes it possible to suppress foamingand strengthen the stirring. That is, the use of a centrifugal stirreris preferable from the viewpoint of rapid desolvation.

The structure of the centrifugal stirrer is a rotating body which formsa flow path by connecting an inlet port near the rotating shaft and adischarge port distant from the rotating shaft. The principle ofstirring is as follows: (1) Stirrer is rotated, (2) Centrifugal forceacts on the flow path, (3) Fluid is discharged in the transversedirection, (4) Vertical flow path becomes a negative pressure, and (5)Negative pressure suction flow is generated in the downward direction.

An M-Revo (registered trademark, manufactured by Medech Co., Ltd.) orthe like may be used as the centrifugal stirrer. A schematic diagram ofM-Revo is shown in FIG. 1 (quoted from the home page of themanufacturer). When M-Revo is rotated, a centrifugal force is generatedat the discharge port (a), and the fluid is discharged in the transversedirection from (a). As a result, a suction force is generated in theinlet port (b), and a tornado-like vortex flow (c) is generated. Anegative pressure is generated in the vertical flow path, a negativepressure suction flow occurs in the downward direction, and a“push→pull” flow is generated. A pulsation (pulse) is transmitted fromthe M-Revo to the stirring flow and the stirring flow propagatesthroughout.

The dimension of the rotor of the centrifugal stirrer is preferably 70to 200 mm, more preferably 80 to 150 mm in diameter, and it ispreferable to have a plurality of (for example, 2 to 10) discharge portsin the circumferential portion. The discharge rate coefficient (totalopening area of the discharge ports×circumferential length), which is anindex of the stirring capacity of the rotor, is preferably 60 cm³ to6,000 cm³, and more preferably 200 cm³ to 2,000 cm³.

The rotation speed of the centrifugal stirrer is preferably 100 to 1,500rpm (rotation/minute), more preferably 200 to 1,000 rpm, and still morepreferably 300 to 800 rpm.

In the case where the radius of the tank is taken to be 1, it ispreferable with respect to the eccentricity that the rotating shaft islocated at a position of 0 (center of tank=0) to 0.8 from the center ofthe tank, but it is necessary that the rotor and the inner wall of thetank are not in contact with each other.

Preferably, in the evaporating step, the gas can be sucked into thecontainer from the central portion of the upper portion of thecontainer, and the gas and the condensed organic solvent can bedischarged from the peripheral portion of the upper portion of thecontainer. As mentioned above, blowing air in the direction from thecenter to the outer periphery is preferable from the viewpoint of rapiddesolvation.

In the present invention, in the step of evaporating an organic solvent,it is preferred that the concentration of an organic solvent containedin an aqueous solution containing liposomes is to be 15 mass % or lesswithin 30 minutes since the start of a step of evaporating the organicsolvent.

A liquid temperature when carrying out the production method of thepresent invention can be appropriately adjusted, but the liquidtemperature at the time of mixing an oil phase and a water phase ispreferably higher than or equal to a phase transition temperature of thelipid to be used. For example, in the case where a lipid having a phasetransition temperature of 35° C. to 40° C. is used, the liquidtemperature is preferably set to 35° C. to 70° C.

The aqueous solution containing the liposomes obtained by the presentinvention may be subjected to post-processing such as centrifugation,ultrafiltration, dialysis, gel filtration, or freeze-drying, for removalof components that had not been included in the liposomes, or adjustmentof a concentration and an osmotic pressure.

Particle sizes of the resulting liposomes can be made uniform by usingdialysis, filtration, extrusion processing, or the like. In the methodfor producing a liposome according to the present invention, it ispreferred that empty liposomes are prepared in a state where a drug isnot loaded, without subjection to extrusion processing. Moreover, if itis desired to separate the drug encapsulated in liposomes from the drugnot encapsulated in liposomes, centrifugation, dialysis, gel filtration,or the like may be employed.

(Apparatus for Producing Liposome)

The apparatus for producing a liposome according to the presentinvention is an apparatus for producing a liposome, including a tank, astirrer, and a jacket, in which the cross-section of the space in thetank has a circular shape, the size of the circle in the cross-sectionis variable depending on the height of the tank, the stirrer is insertedinto the tank from the center of the lid at the top of the tank, thejacket is provided around the tank to control the temperature in thetank, one or more inlet ports for sucking gas into the tank are providedat the central portion of the lid at the top of the tank, and one ormore outlet ports for discharging the gas and condensed organic solventfrom the tank are provided at the periphery of the lid at the top of thetank. The insertion position of the stirrer may be eccentric from thecenter of the tank within the range of eccentricity described inparagraph 0054 of the present specification.

FIG. 2 shows a top view of an example of the configuration of theapparatus for producing a liposome according to the present invention,and FIG. 3 shows a cross-sectional view thereof.

The shape of the tank 1 when viewed from the top is substantially acircular shape, that is, the cross-section of the space in the tank hasa substantially circular shape in which the size of the circlerepresented by the cross-section of the space in the tank may varydepending on the height of the tank. In the example shown in FIG. 3, thesize of the circle of the cross-section in the top half of the tank isalmost constant, whereas the size of the circle of the cross-section inthe bottom half of the tank varies depending on the height of the tank,so that the size of the circle of the cross-section is small for aregion where the height of the tank is low.

A stirrer 15 is inserted into the tank from a center 11 of the lid atthe top of the tank. The stirrer 15 is operated by a stirring means 14.

A jacket 22 for controlling the temperature in the tank is providedaround the tank 1.

Four inlet ports 12 for sucking gas into the tank are provided in thecentral portion of the lid (adjacent region around the center 11) at thetop of the tank 1. Four outlet ports 13 for discharging gas andcondensed organic solvent from the tank are provided in the peripheralportion of the lid (region adjacent to the outer periphery of the lid)at the top of the tank 1.

The reference numeral 21 in FIG. 3 indicates the liquid surface(gas-liquid interface) when the liquid is introduced.

(Drug Loading Step)

The liposome obtained by the method of the present invention canencapsulate a drug. In the drug loading step of encapsulating the drug,in the case of encapsulating a water-soluble drug in the liposomes, thedrug can be encapsulated in the inner water phase of the liposome by amethod of dissolving the drug in an aqueous medium capable of performinghydration and swelling, followed by heating at a temperature higher thanor equal to the phase transition temperature, and sonication orextrusion. A drug may also be encapsulated in an inner water phase bydissolving the drug in the water phase at a time of lipidemulsification.

The drug contained in the liposome may be any water-soluble drug thatcan be encapsulated in liposomes, and specific examples thereof include,but are not limited to, water-soluble materials having a physiologicalactivity or a pharmacological activity such as enzymes, proteins,peptides, nucleic acids (DNA, mRNA, siRNA, miRNA), low-molecular weightcompounds, sugars (oligosaccharides and polysaccharides), polymercompounds, antitumor agents, antimicrobial agents, contrast agents,antioxidants, anti-inflammatory agents, whitening agents, humectants,and hair growing agents. In the case of using a liposome as a carrierfor a drug delivery system, the water-soluble drug is preferably alow-molecular weight compound from the viewpoint of stability.

Specific examples of the water-soluble drug include anticancer agentssuch as an anthracycline-based anticancer agent such as doxorubicin,daunorubicin or epirubicin, a cisplatin-based anticancer agent such ascisplatin or oxaliplatin, a taxane-based anticancer agent such aspaclitaxel or docetaxel, a vinca alkaloid-based anticancer agent such asvincristine or vinblastine, a bleomycin-based anticancer agent such asbleomycin, a sirolimus-based anticancer agent such as sirolimus, and ametabolic antagonist such as methotrexate, fluorouracil, gemcitabine,cytarabine, or pemetrexed. Among these, preferred is a water-solubledrug such as doxorubicin, gemcitabine, or pemetrexed.

The water-soluble drug encapsulated in the liposome is present in adissolved state in the inner water phase of the liposome. Here, withregard to the dissolved state, it is deemed to be encapsulated as adissolved state in a case where the amount of the drug filled withrespect to the volume of the liposome is below the saturation solubilityof the drug in the composition liquid of the inner water phase. Further,even when the amount of the drug filled is above the saturationsolubility of the drug, a case where drug crystals are not observed by acryo-transmission electron microscope (Cryo-TEM) and diffractionpatterns attributable to crystal lattice are not observed by X-raydiffraction (XRD) measurement indicates that most of the drug isdissolved due to acceleration of dissolution by a physicochemicalenvironment created by the lipid membrane, partial incorporation of thedrug into the lipid membrane or the like, and is deemed to beencapsulated as a dissolved state. In addition, a substance encapsulatedby a loading method in which a solid substance is formed inside theliposome and a drug is encapsulated is not in a dissolved state, evenwhen it is a highly water-soluble drug.

The water-soluble drug to be encapsulated in a dissolved statepreferably has a solubility in water of 1 mg/ml or more, and morepreferably a solubility in water of 10 mg/ml or more.

(Sterile Filtration)

In order to formulate an aqueous solution containing liposomes, obtainedby the method for producing a liposome composition according to thepresent invention, into a pharmaceutical composition, it is preferableto carry out sterile filtration. Regarding the filtration method, it ispossible to remove unwanted materials from the aqueous solutioncontaining liposomes by using a hollow fiber membrane, a reverse osmosismembrane, a membrane filter or the like. In the present invention, theaqueous solution containing liposomes is preferably filtered using afilter having a sterile pore size (preferably 0.2 μm sterile filter)although there is no particular limitation. Normally, adsorption oraggregation of liposomes onto a sterile filter may occur in thefiltration step. However, the present invention has unexpected effectssuch as little influence in pressure loss or the like when performingfiltration, since liposomes having a specific average particle size anda uniform particle size distribution are obtained.

To prevent an effect of deformation of liposomes on the average particlesize, the sterile filtration step and the below-described asepticfilling step are preferably carried out at a temperature lower than orequal to the phase transition temperature of the lipids constituting theliposome. For example, in the case where the phase transitiontemperature of the lipid is around 50° C., the sterile filtration stepand the below-described aseptic filling step are carried out at atemperature of preferably about 0° C. to 40° C., and more specificallyabout 5° C. to 30° C.

(Aseptic Filling)

The aqueous solution containing the liposomes obtained after sterilefiltration is preferably aseptically filled for medical applications.Known methods can be applied for aseptic filling. A liposome compositionsuitable for medical applications can be prepared by aseptically fillingthe liposome-containing aqueous solution in a container.

An aqueous solvent, an additive, or the like may be appropriately addedto the aqueous solution containing the liposomes obtained by the presentinvention to thereby prepare a pharmaceutical composition containingliposomes. In connection with the route of administration, thepharmaceutical composition may also contain at least one of a tonicityagent, a stabilizer, an antioxidant, or a pH adjusting agent which ispharmaceutically acceptable.

The tonicity agent is not particularly limited and examples thereofinclude inorganic salts such as sodium chloride, potassium chloride,sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassiumdihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol;and sugars such as glucose, fructose, lactose, and sucrose.

The stabilizer is not particularly limited and examples thereof includesugars such as glycerol, mannitol, sorbitol, lactose, and sucrose.

The antioxidant is not particularly limited and examples thereof includeascorbic acid, uric acid, tocopherol homologues (for example, vitamin E,four tocopherol isomers α, β, γ, and δ), cysteine, and EDTA. Stabilizersand antioxidants may be respectively used alone or in combination of twoor more thereof.

Examples of the pH adjusting agent include sodium hydroxide, citricacid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodiumdihydrogen phosphate, and potassium dihydrogen phosphate.

The pharmaceutical composition may contain an organic solvent, collagen,polyvinyl alcohol, polyvinyl pyrrolidone, a carboxyvinyl polymer, sodiumcarboxymethyl cellulose, sodium polyacrylate, sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin,agar, diglycerol, propylene glycol, polyethylene glycol, vaseline,paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA),mannitol, sorbitol, lactose, PBS, sodium chloride, sugars, abiodegradable polymer, a serum-free medium, each of which ispharmaceutically acceptable, or an additive which is acceptable as apharmaceutical additive.

In particular, in the context of the present invention, thepharmaceutical composition preferably contains ammonium sulfate,L-histidine, purified sucrose, sodium hydroxide, hydrochloric acid, orthe like.

The container in which a pharmaceutical composition is filled is notparticularly limited, and it is preferably made out of a material havinglow oxygen permeability. Examples of the container include a plasticcontainer, a glass container, and a bag made out of a laminate filmhaving an aluminium foil, an aluminium-deposited film, an aluminiumoxide-deposited film, a silicon oxide-deposited film, a polyvinylalcohol, an ethylene-vinyl alcohol copolymer, a polyethyleneterephthalate, a polyethylene naphthalate, a polyvinylidene chloride, orthe like as a gas barrier layer. If necessary, light may be shielded byadopting a bag or the like using a colored glass, an aluminium foil, analuminium-deposited film, or the like.

In the container in which a pharmaceutical composition is filled, inorder to prevent oxidation by oxygen present in the space of thecontainer, it is preferable to replace the gases in the container spaceand drug solution with inert gases such as nitrogen. For example, aninjection solution is bubbled with nitrogen, whereby the filling of theinjection solution into a container can be carried out under a nitrogenatmosphere.

The administration method of a pharmaceutical composition is preferablya parenteral administration. For example, intravenous injections such asintravenous drip, intramuscular injection, intraperitoneal injection,subcutaneous injection, intraocular injection, or intrathecal injectionmay be selected. The specific administration method of a liposomecomposition includes, for example, a syringe and administration byintravenous drip.

The dose of a drug contained in the pharmaceutical composition isusually selected from a range of 0.01 mg to 100 mg/kg body weight/day.However, the liposome composition of the present invention is notlimited to such a dose.

EXAMPLES

Hereinafter, the present invention will be described in detail withreference to Examples. However, the present invention is not limited tosuch Examples.

Example 1

a) Preparation of Equipment

A tank having a structure shown in FIG. 2 and FIG. 3 was prepared.

The tank was equipped with an M-Revo (registered trademark, manufacturedby Medech Co., Ltd.) which was used as a centrifugal stirrer.

The shape of the tank is such that A/B is 4 and C/D is 4 in the casewhere 25.5 kg of water and 7.8 kg of ethanol (about 33.3 L in total) areintroduced.

b) Preparation of Test Solution

As the test solution, 25.5 kg of water and 7.8 kg of ethanol were used.

c) Desolvation

25.5 kg of water and 7.8 kg of ethanol were introduced into the tank.80° C. hot water was circulated in the jacket of the tank such that thetemperature was increased. When the internal temperature of the tankreached 70° C., the evaporation process was started. Stirring with acentrifugal stirrer was started and simultaneously, air at a temperatureof 20° C. was blown at an air flow rate of 4.0 Nm³/min.

The rotor of the centrifugal stirrer has a diameter of 10 cm and has sixdischarge ports with a diameter of 1 cm in the circumferential portion.The rotation speed of the centrifugal stirrer was set to 600 rpm. Theamount of eccentricity was such that the rotating shaft was positioned65 mm external from the center of the tank. The discharge ratecoefficient (total opening area of discharge ports×circumferentiallength), which is an index of the stirring capacity of the rotor, was591 cm³.

Air was sucked from the inlet port at the top of the tank and dischargedfrom the outlet port at the top of the tank to thereby blow air in adirection from the center to the outer periphery.

After 10 minutes, the evaporating step was completed, and the liquid wasrecovered from the tank. The mass of the recovered liquid was measuredand the residual concentration of ethanol was measured by gaschromatography to thereby calculate the evaporation amount of water andethanol during the evaporation process. The results of the abovecalculation are shown in Table 1. The dew point (calculated value) was33° C.

Comparative Example 1

The evaporation process was carried out in the same manner as in Example1, except that the blowing air temperature was set to 50° C. by aheating device. As the heating device, a heat exchanger in which hotwater passes through the primary side and a carrier (air or nitrogen)passes through the secondary side was used.

The calculated results of the evaporation amount of water and theevaporation amount of ethanol are shown in Table 1. The dew point(calculated value) was 31° C.

Comparative Example 2

The evaporation process was carried out in the same manner as in Example1, except that the air flow rate was set to 12 Nm³/min.

The calculated results of the evaporation amount of water and theevaporation amount of ethanol are shown in Table 1. The dew point(calculated value) was 14° C.

Example 2

The evaporation process was carried out in the same manner as in Example1, except that the shape of the tank was such that A/B was 1 and C/D was4 in the case where 25.5 kg of water and 7.8 kg of ethanol (about 33.3 Lin total) were introduced.

The calculated results of the evaporation amount of water and theevaporation amount of ethanol are shown in Table 1. The dew point(calculated value) was 26° C.

Example 3

The evaporation process was carried out in the same manner as in Example1, except that the shape of the tank was such that A/B was 4 and C/D was2 in the case where 25.5 kg of water and 7.8 kg of ethanol (about 33.3 Lin total) were introduced.

The calculated results of the evaporation amount of water and theevaporation amount of ethanol are shown in Table 1. The dew point(calculated value) was 27° C.

Example 4

The evaporation process was carried out in the same manner as in Example1, except that the direction of air blowing was set to one direction.

The calculated results of the evaporation amount of water and theevaporation amount of ethanol are shown in Table 1. The dew point(calculated value) was 27° C.

Example 5

The evaporation process was carried out in the same manner as in Example1, except that a paddle type stirrer (blade diameter: 100 mm) was usedinstead of the centrifugal stirrer and the rotation speed was set to 350rpm.

The calculated results of the evaporation amount of water and theevaporation amount of ethanol are shown in Table 1. The dew point(calculated value) was 30° C.

In the foregoing Examples and Comparative Examples, the rate ofdesolvation was determined according to the following standards. RatingsA to C are acceptable levels.

A: The evaporation amount of ethanol was 100 g/min or more.

B: The evaporation amount of ethanol was 70 g/min or more and less than100 g/min.

C: The evaporation amount of ethanol was 50 g/min or more and less than70 g/min.

The dew point of each example was calculated by the following method.

1) Vapor partial pressures of water and ethanol were calculated from theactual values of evaporation amount (water), evaporation amount(ethanol) and air flow rate using the state equation of gas, and thetotal of the calculated vapor partial pressures was taken as the totalpressure of the vapor.

2) From the total vapor pressure calculated in 1) and the introducedmole fraction of the liquid composition, the dew point in the state wascalculated using Antoine equation and Wilson equation in vapor-liquidequilibrium.

The details of the method for calculating vapor-liquid equilibrium aredescribed in the following literatures.

“Theory and Calculation of Phase Equilibrium for the Separation”, pp. 75to 76, written by Shuzo Ohe (Kodansha Ltd., 2012)

“Vapor-Liquid Equilibrium Data Collection”, p 216, written by Shuzo Ohe(Kodansha Ltd., 1988).

TABLE 1 Comparative Comparative Example 1 Example 1 Example 2 Example 2Example 3 Example 4 Example 5 Heating device No Yes No No No No NoTank-shaped liquid 4 4 4 1 4 4 4 part A/B Tank-shaped gas flow 4 4 4 4 24 4 part C/D Air blowing From center From center to From center to Fromcenter to From center to One direction From center to to outer outerperiphery outer periphery outer outer outer periphery peripheryperiphery periphery Stirrer Centrifugal Centrifugal CentrifugalCentrifugal Centrifugal Centrifugal Paddle type Air flow rate Nm³/min4.0 4.0 12 4.0 4.0 4.0 4.0 Blowing air 20 50 20 20 20 20 20 temperature° C. Evaporation amount 182 187 185 127 135 130 85 (water) g/minEvaporation amount 106 110 107 75 78 76 51 (ethanol) g/min Dew point °C. 33 31 14 26 27 27 30 Desolvation rate A A A B B B C Capitalinvestment Acceptable Non-acceptable Non-acceptable AcceptableAcceptable Acceptable Acceptable burden

As can be seen from Table 1, in Examples 1 to 5 of the presentinvention, it was possible to achieve the desolvation rate that could betolerated under the conditions without a capital investment burden.

Example 6

a) Preparation of Emulsification Equipment

The emulsification equipment was prepared by combining a circulationpath 32 (the capacity of the circulation path is 6 L) including anintermittent jet flow generating type emulsifying apparatus 33 having arotating part with a slit outer diameter of 90 mm (CLEARMIX (registeredtrademark) manufactured by M Technique Co., Ltd.), an emulsifying tank31 with a full-water capacity of 26 L, a defoaming tank 34, and acirculating pump 35 (direct-acting diaphragm pump model EJL1500S12manufactured by Nikkiso-LEWA GmbH) as shown in FIG. 4. The capacity ofthe circulation path is the capacity of the entire circulation pathincluding the defoaming tank.

b) Preparation of Oil Phase

Hydrogenated soybean phosphatidylcholine, cholesterol andN-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt(hereinafter referred to as DSPE-PEG) were mixed to a molar ratio of57/38/5, and then an organic solvent (ethanol) was added thereto,followed by warming to 70° C. and dissolution of the lipids to preparean oil phase. Here, the oil phase was prepared such that the lipidconcentration in the oil phase was 70 mmol/L.

c) Preparation of Water Phase

177 mmol/L of an aqueous ammonium sulfate solution was prepared and wasthen prepared as a water phase in an emulsifying tank.

d) Emulsification

The water phase prepared in c) was heated to 70° C. The oil phaseprepared in b) was added so that the volume ratio of water phase/oilphase=8/3 was obtained while operating an emulsifying apparatus at acircumferential speed of 15 m/s and operating a circulation pump at aflow rate of 20 L/min. The total amount of the liquid after the additionwas 32 L. After the emulsifying tank was fully hydrated, the overflowed6 L circulated through the circulation path including the defoamingtank. The internal pressure of the emulsifying tank was set to 30 kPa.In this state, the rotation of the emulsifying apparatus was increasedto a circumferential speed of 30 m/sec, followed by stirring for 30minutes.

e) Desolvation

The emulsion prepared in d) was quickly transferred to a desolvationtank (which is the same as that used in Example 1). 80° C. hot water wascirculated in the jacket of the tank, so that the evaporation processwas started as the temperature warmed. At the same time as starting thestirring in the centrifugal stirrer (the same conditions as in Example1), 20° C. air was blown at a rate of 4.0 Nm³/min. After 10 minutes, thetemperature of the hot water in the jacket was modified to 60° C. andthe air flow rate was changed to 3.0 Nm³/min. After 60 minutes from thestart, the rotation speed of the centrifugal stirrer was changed to 450rpm and the air flow rate was changed to 1.4 Nm³/min. After 4 hours fromthe start, the rotation speed of the centrifugal stirrer was changed to300 rpm. The evaporation process was completed after 6 hours from thestart, and the liposome liquid was recovered from the tank. The dewpoint (calculated value) was 33° C. at the start of desolvation.

Example 7

The liposome liquid was prepared and recovered in the same manner as inExample 6, except that a paddle type stirrer (blade diameter: 100 mm)was used instead of the centrifugal stirrer and the rotation speed wasset to 350 rpm at the start of desolvation and 150 rpm after 60 minutesfrom the start of the desolvation. The dew point (calculated value) was33° C. at the start of desolvation.

(Measurement of Average Particle Size of Liposomes by Dynamic LightScattering Method)

The liposome liquids prepared in Examples 6 and 7 were 40-fold dilutedwith pure water to obtain samples for measuring an average particlesize. The average particle size of the samples for measuring an averageparticle size was measured in terms of volume average particle sizeusing a particle size analyzer FPAR-1000AS (manufactured by OtsukaElectronics Co., Ltd.).

The measurement results are shown in Table 2. As shown in Table 2,although it is possible to produce liposomes using any one of acentrifugal stirrer or a paddle type stirrer, it was found that the useof a centrifugal stirrer is preferable since liposomes having a smalleraverage particle size can be produced as in Example 6 using acentrifugal stirrer. Because Example 7 employs a lipid (DSPE-PEG) whichreadily generates bubbles, the amount of bubbles increases and thereforethe desolvation tends to be delayed in the case of using a paddle typestirrer.

TABLE 2 Example 6 Example 7 Heating device No No Tank-shaped liquid partA/B 4 4 Tank-shaped gas flow part C/D 4 4 Air blowing From center Fromcenter to outer to outer periphery periphery Stirrer Centrifugal Paddletype Air flow rate Nm³/min 4.0 to 2.0 4.0 to 2.0 Blowing air temperature° C. 20 20 Dew point ° C. (at start of 33 33 desolvation) Averageparticle size nm 59 81

Example 8

Preparation of Drug-Encapsulated Liposomes

With respect to the non-drug encapsulated liposomes prepared in Example6, the lipid concentration of the liposome liquid was concentrated to arange of 120 to 150 mmol/L by tangential flow filtration, while theouter water phase of the liposome was replaced with a 0.09 mass %aqueous sodium chloride solution. A drug solution prepared by heating todissolve gemcitabine hydrochloride in a phosphate buffer solution wasadded thereto. The mixture was heated at about 70° C. for 30 minutes andthen cooled to room temperature. Thereafter, the drug-liposome mixturewas filtered through a sterilization filter having a pore size of 0.2μm. Further, the drug-liposome mixture was purified by dialysis againsta 9.4 mass % sucrose/10 mmol/L-histidine aqueous solution by tangentialflow filtration to remove non-encapsulated gemcitabine. This wasfollowed by filtration of liposomes through a sterilizing filter havinga pore size of 0.2 μm to obtain a solution of sterilegemcitabine-encapsulated liposomes having an average particle size of 69nm. According to the present invention, it was confirmed that liposomeshaving a single dispersion peak and a small average particle size of 100nm or less usable for medical applications can be produced.

INDUSTRIAL APPLICABILITY

According to the present invention, liposomes can be produced on largescale production. The liposomes produced by the present invention can beapplied to medicines, cosmetics, foods, or the like, and areparticularly useful for medical applications.

EXPLANATION OF REFERENCES

-   -   1: tank    -   11: center    -   12: inlet port    -   13: outlet port    -   14: stirring means    -   15: stirrer    -   21: liquid surface    -   22: jacket    -   31: emulsifying tank    -   32: circulation path    -   33: emulsifying apparatus    -   34: defoaming tank    -   35: pump

What is claimed is:
 1. A system for producing a liposome, comprisingemulsification equipment and desolvation equipment, wherein thedesolvation equipment comprises a tank; a stirrer; and a jacket, whereinthe cross-section of the space in the tank has a circular shape, thesize of the circle in the cross-section is variable depending on theheight of the tank and the diameter of the cross-section of the space inthe tank is longer than the height of the tank, the stirrer is insertedinto the tank from the center of a lid at the top of the tank, thejacket is provided around the tank to control the temperature in thetank, one or more inlet ports for sucking gas into the tank are providedat the central portion of the lid at the top of the tank, and one ormore outlet ports for discharging the gas and condensed organic solventfrom the tank are provided at a peripheral portion of a gas space abovea liquid in the tank comprising an organic solvent or organic solvents,and wherein the emulsification equipment comprises an emulsifying tankand an emulsifying apparatus.
 2. The system for producing a liposomeaccording to claim 1, wherein the capacity of the tank is within a rangeof 10 L or more and 100 L or less.
 3. The system for producing aliposome according to claim 1, wherein a stirring step and anevaporating step are carried out in the tank, and the ratio A/B oflength A of the widest portion of a liquid horizontal plane of theliquid comprising the organic solvent in the tank at the start of theevaporating step to length B of a deepest portion of the liquid depth is2 or more.
 4. The system for producing a liposome according to claim 2,wherein a stirring step and an evaporating step are carried out in thetank, and the ratio A/B of length A of the widest portion of a liquidhorizontal plane of the liquid comprising the organic solvent in thetank at the start of the evaporating step to length B of a deepestportion of the liquid depth is 2 or more.
 5. The system for producing aliposome according to claim 1, wherein a stirring step and anevaporating step are carried out in the tank, and the ratio C/D oflength C of the widest portion of a horizontal plane of the gas space inthe tank at the start of the evaporating step to length D of the longestportion of the height of the gas space is 3 or more, wherein the gasspace is a space occupied by the gas flowing in a horizontal directionon a liquid surface.
 6. The system for producing a liposome according toclaim 2, wherein a stirring step and an evaporating step are carried outin the tank, and the ratio C/D of length C of the widest portion of ahorizontal plane of the gas space in the tank at the start of theevaporating step to length D of the longest portion of the height of thegas space is 3 or more, wherein the gas space is a space occupied by thegas flowing in a horizontal direction on a liquid surface.
 7. The systemfor producing a liposome according to claim 3, wherein the stirring stepand the evaporating step are carried out in the tank, and the ratio C/Dof length C of the widest portion of the horizontal plane of the gasspace in the tank at the start of the evaporating step to length D ofthe longest portion of the height of the gas space is 3 or more, whereinthe gas space is a space occupied by the gas flowing in a horizontaldirection on a liquid surface.
 8. The system for producing a liposomeaccording to claim 4, wherein the stirring step and the evaporating stepare carried out in the tank, and the ratio C/D of length C of the widestportion of the horizontal plane of the gas space in the tank at thestart of the evaporating step to length D of the longest portion of theheight of the gas space is 3 or more, wherein the gas space is a spaceoccupied by the gas flowing in a horizontal direction on a liquidsurface.
 9. The system for producing a liposome according to claim 1,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 10. The system for producing a liposome according to claim 2,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 11. The system for producing a liposome according to claim 3,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 12. The system for producing a liposome according to claim 4,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 13. The system for producing a liposome according to claim 5,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 14. The system for producing a liposome according to claim 6,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 15. The system for producing a liposome according to claim 7,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 16. The system for producing a liposome according to claim 8,wherein the stirrer has 2 to 10 discharge ports, a discharge ratecoefficient indicated by a total opening area of the discharge portsmultiplied by a circumferential length of a rotor of the stirrer is 60cm³ to 6,000 cm³, and the rotation speed of the stirrer is 100 to 1,500rpm.
 17. The system for producing a liposome according to claim 1,wherein the one or more inlet ports comprises one to four inlet portsfor sucking gas into the tank are provided at a central portion of thelid at the top of the tank.
 18. The system for producing a liposomeaccording to claim 1, wherein the one or more outlet ports comprises oneto four outlet ports for discharging the gas and condensed organicsolvent from the tank are provided at a peripheral portion of a gasspace above a liquid surface of a liquid comprising the organic solventis contained in the tank.
 19. The system for producing a liposomeaccording to claim 1, wherein the emulsification equipment furthercomprises a defoaming tank and a circulation path.
 20. The system forproducing a liposome according to claim 19, wherein the emulsifying tankis connected with the defoaming tank through the circulation path.